US4956449A - Immunologically active synthetic peptides useful for preparing an antimalarial vaccine - Google Patents

Immunologically active synthetic peptides useful for preparing an antimalarial vaccine Download PDF

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US4956449A
US4956449A US07/276,699 US27669988A US4956449A US 4956449 A US4956449 A US 4956449A US 27669988 A US27669988 A US 27669988A US 4956449 A US4956449 A US 4956449A
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asn
process according
pro
tetrapeptide
formula
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Antonio S. Verdini
Fabio Bonelli
Antonello Pessi
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Eni Tecnologie SpA
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Eniricerche SpA
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Assigned to ENIRICERCHE S.P.A. reassignment ENIRICERCHE S.P.A. ASSIGNMENT OF ASSIGNORS INTEREST. Assignors: BONELLI, FABIO, PESSI, ANTONELLO, VERDINI, ANTONIO S.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
    • C07K16/205—Plasmodium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to new immunologically active synthetic peptides useful in the malaria sector.
  • the invention relates to synthetic peptides which are able to induce in mammals a high-count antibody response specific not only towards themselves but also towards the immunodominant epitope of the circumsporozoitic protein of Plasmodium falciparum.
  • the invention also relates to the use of said peptides for preparing antimalarial vaccines and diagnostic kits for detecting malarial antiparasite antibodies in clinical samples.
  • Malaria caused by a protozoon of the genus Plasmodium, currently represents one of the most serious parasite illnesses of man.
  • the infection begins in man with the introduction of sporozoites by the mosquito, which localize rapidly in the hepatic cells. Here each sporozoite splits into 20,000 or more merozoites, each of which after leaving the hepatic cell is able to infect a red corpuscle. Within the erythrocyte the parasite reproduces asexually from rings to schizonts.
  • the mature schizont contains individual merozoites able to invade other red corpuscles.
  • malarial parasites develop by a multi-stage cycle and present the host with a very large number of antigenic components, each parasite development form containing different stage-specific antigens.
  • Plasmodium sporozoites were of particular interest because the preparation of an antisporozoite vaccine, if completely effective, would prevent the development of the plasmodium in the host and would therefore induce sterile immunity.
  • circumsporozoitic protein This protein, known as circumsporozoitic protein or CS, entirely covers the sporozoite surface and induces a specific antibody response which confers protection against malarial infections.
  • the primary structure deduced from the gene nucleotide sequence shows that the immunodominant epitope in the CS protein consists of the tetrapeptide Asn-Ala-Asn-Pro (NANP) repeated 37 times plus 4 Asn-Val-Asp-Pro (NVDP) quadruplets.
  • NANP tetrapeptide Asn-Ala-Asn-Pro
  • NVDP Asn-Val-Asp-Pro
  • Copending U.S. patent application Ser. No. 850,135 describes and claims sequential polypeptides consisting of the tetrapeptide (NANP) repeated n times, preferably 40 times, and obtained by polycondensation. Said polypeptides are recognised by antisporozoite antibodies present in the serum of individuals exposed to malarial infection and are able to induce in animals the formation of anti-(NANP) n antibodies even when not conjugated to a protein support. However said polypeptides when inoculated into mice induce an immune response which is subject to genetic restriction. In fact, it has been observed that only mice whose genetic makeup comprises the gene I-A b (responder) recognise the T epitope contained in the repeated sequence of the CS protein and are therefore able to produce an anti-(NANP) n antibody response.
  • I-A b responder
  • New sequential peptides consisting of at least two repeated (Ala-Asn-Asn-Pro) (ANNP) units have now been synthesized which are able to induce in-vivo a specific antibody response for said peptides and for the immunodominant epitope of the CS protein.
  • An object of the present invention is therefore to provide immunologically active sequential peptides able to induce in mammals a high-count antibody response useful in the malaria sector.
  • a further object of the present invention is to provide a process for preparing said synthetic peptides.
  • a further object of the present invention is the use of said sequential peptides for preparing diagnostic kits for the determination of antisporozoite antibodies in human clinical samples.
  • peptides according to the present invention can be defined by the general formula:
  • Asn is asparagine, Pro is proline and Ala is alanine, and n is between 2 and 100.
  • said peptides can be prepared by a process comprising:
  • X is an acid-labile protector group
  • the tetrapeptide (II) is prepared by homogeneous phase condensation using one of the known general methods.
  • the procedure is to dissolve the amino acids, suitably protected at the reactive functions, in an inert (unreactive) organic solvent, in the presence of condensation agents.
  • Suitable organic solvents for this purpose are chosen from chlorinated aliphatic hydrocarbons, aliphatic aldehydes and alkyl esters. Specific examples of said solvents are N,N-dimethylformamide, chloroform, ethylacetate and tetrahydrofuran.
  • Protector groups for the amino functions are generally chosen from those which can be removed by acid hydrolysis (acid-labile). Particularly preferred of these is tert-butyloxycarbonyl (Boc) which is removable under mild hydrolysis conditions.
  • the temperature at which the condensation reaction is carried out varies generally between -10° and 40° C. and the corresponding time is that required for the reaction to go to completion or substantial completion.
  • stage (b) of the process according to the present invention the tetrapeptide (II) protected at the terminal amino group is activated by reaction with a phenol derivative to form the active ester of said tetrapeptide at the terminal Pro carboxyl:
  • Halogenated phenol derivatives usable in the process of the present invention are the fluorinated or chlorinated derivatives. Particularly suitable for this purpose are pentachlorophenol, trichlorophenol and pentafluorophenol.
  • the activation reaction at the Pro carboxyl group is conducted by bringing the tetrapeptide (II) into contact with the halogenated phenol derivative in a molar ratio equal or approximately equal to 1, in an organic solvent liquid environment, operating at a temperature of between -10° C. and 40° C.
  • the reaction is preferably conducted at ambient (20°-25° C.) or near ambient temperature.
  • Suitable solvents for this purpose are aprotic solvents such as ethyl acetate, or aliphatic hydrocarbons, or dimethylformamide.
  • the solution obtained is cooled to about 0° C. and a condensation agent is then added, the molar ratio of condensation agent to one or other of the starting reagents being equal or approximately equal to 1.
  • the condensation agent used is preferably dicyclohexylcarbodiimide (DCCI).
  • the solution thus obtained is maintained at a temperature of between -10° C. and 40° C. for a time of between 4 hours and 15 minutes.
  • the reaction mixture On termination of the reaction, the reaction mixture is separated from the dicyclohexylurea (DCU) which forms and the solvent evaporated.
  • DCU dicyclohexylurea
  • stage (c) of the process according to the invention the protector group is removed from the amino terminal of the tetrapeptide (III) by acid hydrolysis.
  • the reaction is conducted using trifluoroacetic acid or a solution of hydrochloric acid in ethyl acetate operating at ambient temperature (20°-25° C.) for a time of about 1 hour.
  • Organic bases suitable for this purpose are tertiary alkylamines in which the alkyl group is formed from 1-4 carbon atoms. Triethylamine is particularly preferred.
  • the polycondensation reaction is conducted in an organic solvent chosen from dimethylsulphoxide, dimethylformamide and hexamethylphosphoramide, operating at a temperature of between -10° C. and 40° C. for a time of between 4 days and 24 hours. In practice the operation is carried out at ambient or near ambient temperature, and in this case the time required for the reaction to go to completion or substantial completion is of the order of 96 hours.
  • an organic solvent chosen from dimethylsulphoxide, dimethylformamide and hexamethylphosphoramide
  • the dried product is then dissolved in a water/dioxane solution and lyophilized.
  • the lyophilized product consisting of a mixture of polypeptides of different molecular weight, can either be used as such for preparing antimalarial vaccines and diagnostic kits or be fractionated by known general methods to obtain polypeptides with a more restricted molecular weight (MW) distribution (stage e).
  • the lyophilized product is fractionated by chromatography in a Sephadex® G-50 column at a temperature of 20°-25° C. eluting with 0.1M acetic acid at a throughput of 36 ml/hour.
  • the peptide of formula (I) can be prepared by a process using condensation agents able to directly polymerize the tetrapeptide H-Ala-Asn-Asn-Pro-OH.
  • condensation agents able to directly polymerize the tetrapeptide H-Ala-Asn-Asn-Pro-OH.
  • N,N'-phenylphosphono bis[2(3H)-benzothiazolone] is particularly preferred.
  • the tetrapeptide with its amino and carboxylic ends unprotected is prepared from the derivative (II) by releasing the protector group X by acidolysis and then dissolved in N-methylpyrrolidone and polymerized with the aforesaid condensation agent in the presence of trimethylamine or N-methylmorpholine.
  • the polymerization can be conducted directly in triethylamine or N-methylmorpholine.
  • the result of inverting the Asn and Ala amino acids in the repeating CS sequence is to stabilize a conformation at the local beta turn chain level of type I.
  • the native structure can assume this conformation, even though for a short time and in equilibrium with others, as demonstrated by chemical and physical studies on the synthetic polypeptide (NANP) 40 .
  • Said peptides are particularly useful for the purposes of the present invention.
  • Particularly suitable are the peptides (ANNP) 11 which in experimental animals are found to be potent immunogens.
  • the antibodies produced recognise not only the synthetic antigen (ANNP) 11 but also the antigen (NANP) 40 .
  • the cooling bath is removed and the reaction is allowed to proceed for about 15 hours at ambient temperature.
  • the dicyclohexylurea which forms is filtered off and the filtrate evaporated to dryness.
  • the solid residue is ground with Et 2 O and then suspended in water (1 hour).
  • the solid residue which remains after sedimenting the aqueous suspension is carefully washed with Et 2 O, while the aqueous solution is brought into contact with a mixed bed of ion exchange resin.
  • the resin is recovered by filtration, washed with water and the solution lyophilized. Chromatographic checks by high-performance reverse phase chromatography indicate that both the solid isolated from the water suspension and that recovered after lyophilization are the same compound and impurities are not present.
  • the product identity is established by n.m.r. analysis and mass spectrometry.
  • the polymer is isolated by precipitation from the reaction mixture diluted with water by adding 40 ml of ethanol.
  • the flocky precipitate is allowed to sediment for some hours and then recovered by decanting the supernatant liquid.
  • the solid is dissolved in water and then lyophilized, taken up in 5 ml of 0.1M acetic acid and, after eliminating a small quantity of settled material, the liquid solution is chromatographed by elution in a column of fine Sephadex G-50 with 0.1M acetic acid.
  • the molecular weight of three different polymer fractions is determined by exclusion chromatography on 5M agarose in 6M guanidine chloride, with reference to the elution positions of polypeptides of known molecular weight.
  • the molecular weight of the least retained fraction was equal to 4,500 (ANNP) 11 .
  • the capacity of (Ala-Asn-Asn-Pro) 11 [(ANNP) 11 ] to induce an antibody response in experimental animals was tested by immunising male rabbits aged 5 weeks with the synthetic peptide.
  • the specificity of the formed antibodies is determined by the ELISA (enzyme-linked-surface-immuno-assay) immunoenzymatic assay using both (Ala-Asn-Asn-Pro) 11 and (Asn-Ala-Asn-Pro) 40 as antigens to reproduce the immunodominant epitope of the CS protein of Plasmodium falciparum, synthesized as described in the U.S. patent application Ser. No. 850,135 filed on Apr. 10, 1986.
  • the organic phase is separated from the solution and anhydrified with about 10 g of anhydrous MgSO 4 .
  • step (a) 117 g (28 mmole) of Boc-Asn-Pro-OBz obtained according to step (a) is added to 200 ml of a BtOAc solution containing 4N HCl.
  • the stirred solution is kept at room temperature (20°-25° C.) for about 1 hour.
  • the condensation reaction is carried out by keeping the solution at a temperature of 0° C. for a period of time of about 2 hours.
  • step (a) is followed.
  • the stirred solution is kept at 0° C. for 1 hour and then at room temperature for a period of about 1 hour.
  • the solvent is evaporated under vacuum and the oily residue is tritured with diethylether until a white solid is obtained.
  • the product is suspended in 2 ml of dimethylsulphoxide and the mixture is stirred to obtain a solution.
  • 300 ml of triethylamine is added in small proportions.
  • the solution is kept at room temperature for 24 hours and, after addition of 100 ml of triethylamine, for another 48 hours.
  • the solution is added dropwise, for a period of about 5 minutes, to 350 ml of anhydrous ethanol kept under mild stirring.
  • the obtained precipitate was removed from the suspension by filtration and dried under vacuum.
  • the product is divided into fractions of about 30 mg each, each fraction is dissolved in 1 ml of 0.1N acetic acid and subjected to chromatography separation.
  • a 2.5 ⁇ 80 mm Sephadex® G25 FINE -(Pharmacia Upsala) column is used at a temperature of 20°-25° C. and a flow rate of 0.5 ml/minute.
  • the fractions are collected at equal ranges of 12 minutes.
  • the fractions from 26 to 39 are gathered together and lyophilized.
  • Said polymer is then fractioned by gel chromatography operating in the same way as above using a column filled with Sephadex® G50.
  • the molecular weight of the fractions having n from 37 to 41 is confirmed by chromatography on a 1.5 ⁇ 1000 cm column filled with BIOGEL® A-5M (BIORAD) equilibrated with 6M guanidinium chloride using as internal calibration standards albumine (MW 45,000), myoglobin (MW 18,000), trypsin (MW 8,000) and tryptophan (MW 200).
  • the operation is performed at room temperature with a flow rate of 2.5 ml/hour and collecting the fractions every hour.
  • the rabbits (6) are inoculated intramuscularly (1 inoculation) and subcutaneously (4 inoculations) in accordance with the following scheme: 3 rabbits with 1 ml of pH 7.8 phosphate buffered saline (PBS) containing 1 mg of (ANNP) 11 +1 ml of complete Freud's adjuvant (CFA) and 3 rabbits (controls) with 1 ml of PBS+1 ml of CFA.
  • PBS pH 7.8 phosphate buffered saline
  • NFA complete Freud's adjuvant
  • the sera of the treated animals is withdrawn at days 0, 20, 34 and 48 and analysed by the ELISA to quantify the antibodies formed and to test their specificity.
  • NANP synthetic antigens
  • NRP synthetic antigens
  • the plates are then washed 3 times with PBS-Tween (0.05% Tween 20 v/v, pH 7.4) and the aspecifically bound sites are blocked by incubation at ambient temperature for 1 hour with PBS-Tween-1% (w/v) powdered milk.
  • Scalar dilutions of rabbit serum in 100 ⁇ l of PBS-1% powdered milk are prepared, and 50 ⁇ l of each dilution are inoculated into the wells in the microplates and incubated at ambient temperature for 1 hour.
  • the plates are incubated at ambient temperature for 1 hour and then washed 3 times with PBS-Tween.
  • the synthetic peptide (ANNP) 11 is seen to be a powerful immunogen in experimental animals, and able to induce a high-count antibody response not only against itself but also against the synthetic antigen (NANP) 40 .

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US07/276,699 1987-12-11 1988-11-28 Immunologically active synthetic peptides useful for preparing an antimalarial vaccine Expired - Fee Related US4956449A (en)

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IT22948A/87 1987-12-11
IT8722948A IT1233419B (it) 1987-12-11 1987-12-11 Peptidi sintetici immunologicamente attivi utili per la preparazione di un vaccino antimalaria

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EP (1) EP0320034B1 (de)
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CA (1) CA1316632C (de)
DE (1) DE3851310T2 (de)
ES (1) ES2058240T3 (de)
IT (1) IT1233419B (de)
OA (1) OA08974A (de)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6828416B1 (en) 1998-08-21 2004-12-07 The United States Of America As Represented By The Department Of Health And Human Services Recombinant multivalent malarial vaccine against Plasmodium falciparum

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0378881B1 (de) * 1989-01-17 1993-06-09 ENIRICERCHE S.p.A. Synthetische Peptide und deren Verwendung als allgemeine Träger für die Herstellung von immunogenischen Konjugaten, die für die Entwicklung von synthetischen Impfstoffen geeignet sind
IT1237764B (it) * 1989-11-10 1993-06-17 Eniricerche Spa Peptidi sintetici utili come carriers universali per la preparazione di coniugati immunogenici e loro impiego per lo sviluppo di vaccini sintetici.

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3849551A (en) * 1972-09-28 1974-11-19 Antonio L D Bcg as adjuvant in malaria vaccination
US4416872A (en) * 1982-03-17 1983-11-22 The United States Of America As Represented By The Secretary Of The Army Treatment of malaria with liposomes containing 8-aminoquinoline derivatives and glycoconjugates
US4466917A (en) * 1981-02-12 1984-08-21 New York University Malaria vaccine
WO1986005790A1 (en) * 1985-03-28 1986-10-09 New York University Immunogenic antigen-carrier protein conjugate for use in a vaccine against malaria
US4643896A (en) * 1983-06-10 1987-02-17 Otsuka Pharmaceutical Co., Ltd. Malaria associated antigen and preparing process thereof
US4693994A (en) * 1985-11-19 1987-09-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Protective synthetic peptide against malaria and encoding gene
US4707357A (en) * 1984-06-26 1987-11-17 The United States Of America As Represented By The Secretary Of The Army Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
US4710377A (en) * 1983-08-19 1987-12-01 American Cyanamid Company Antigens and monoclonal antibodies reactive against sporozoites of Eimeria spp.
US4722840A (en) * 1984-09-12 1988-02-02 Chiron Corporation Hybrid particle immunogens
US4735799A (en) * 1987-01-14 1988-04-05 Patarroyo Manuel E Malaria vaccine
US4746612A (en) * 1984-10-12 1988-05-24 Scripps Clinic & Research Foundation Aotus interspecies hybridomas and monoclonal receptors produced thereby
US4767622A (en) * 1983-08-19 1988-08-30 University Of Illinois Method and materials for development of immunological responses protective against malarial infection
US4769235A (en) * 1984-01-27 1988-09-06 New York University Immunodominant epitope of the circumsporozoite surface protein

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3849551A (en) * 1972-09-28 1974-11-19 Antonio L D Bcg as adjuvant in malaria vaccination
US4466917A (en) * 1981-02-12 1984-08-21 New York University Malaria vaccine
US4416872A (en) * 1982-03-17 1983-11-22 The United States Of America As Represented By The Secretary Of The Army Treatment of malaria with liposomes containing 8-aminoquinoline derivatives and glycoconjugates
US4643896A (en) * 1983-06-10 1987-02-17 Otsuka Pharmaceutical Co., Ltd. Malaria associated antigen and preparing process thereof
US4710377A (en) * 1983-08-19 1987-12-01 American Cyanamid Company Antigens and monoclonal antibodies reactive against sporozoites of Eimeria spp.
US4767622A (en) * 1983-08-19 1988-08-30 University Of Illinois Method and materials for development of immunological responses protective against malarial infection
US4769235A (en) * 1984-01-27 1988-09-06 New York University Immunodominant epitope of the circumsporozoite surface protein
US4707357A (en) * 1984-06-26 1987-11-17 The United States Of America As Represented By The Secretary Of The Army Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
US4722840A (en) * 1984-09-12 1988-02-02 Chiron Corporation Hybrid particle immunogens
US4746612A (en) * 1984-10-12 1988-05-24 Scripps Clinic & Research Foundation Aotus interspecies hybridomas and monoclonal receptors produced thereby
WO1986005790A1 (en) * 1985-03-28 1986-10-09 New York University Immunogenic antigen-carrier protein conjugate for use in a vaccine against malaria
US4693994A (en) * 1985-11-19 1987-09-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Protective synthetic peptide against malaria and encoding gene
US4735799A (en) * 1987-01-14 1988-04-05 Patarroyo Manuel E Malaria vaccine

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Proceedings of the National Academy of Sciences of the United States America, vol. 84, No. 13, Jul. 1987, pp. 4470 4474. *
Proceedings of the National Academy of Sciences of the United States America, vol. 84, No. 13, Jul. 1987, pp. 4470-4474.
Proceedings of the National Academy of Sciences of the United States of America, vol. 85, No. 4, Feb. 1988, pp. 1199 1203. *
Proceedings of the National Academy of Sciences of the United States of America, vol. 85, No. 4, Feb. 1988, pp. 1199-1203.

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6828416B1 (en) 1998-08-21 2004-12-07 The United States Of America As Represented By The Department Of Health And Human Services Recombinant multivalent malarial vaccine against Plasmodium falciparum

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OA08974A (en) 1990-11-30
DE3851310T2 (de) 1995-02-16
CA1316632C (en) 1993-04-20
EP0320034A2 (de) 1989-06-14
EP0320034B1 (de) 1994-08-31
EP0320034A3 (de) 1991-01-09
ATE110741T1 (de) 1994-09-15
DE3851310D1 (de) 1994-10-06
IT8722948A0 (it) 1987-12-11
ZA888580B (en) 1989-08-30
ES2058240T3 (es) 1994-11-01
IT1233419B (it) 1992-03-31

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